1. INTRODUCTION:

Globally, millions of blood products are transfused annually.¹ Blood transfusion can save patients undergoing surgery or with congenital or acquired illnesses, but it can also cause infectious and noninfectious issues. Erythrocyte, platelet, and fresh frozen plasma (FFP) concentrates are the most often transfused blood products.^2-5 Red blood cell (RBC) transfusions cure bleeding and increase tissue oxygen supply, whereas platelet transfusions prevent hemorrhage in individuals with thrombocytopenia or platelet function problems. However, fresh frozen plasma infusion can reverse anticoagulant effects.⁵ Health authorities strictly regulate blood product collection, processing, production, and storage in blood banks to ensure safety and purity.^6-9 RBC preservation in blood banks is a life-saving therapeutic intervention globally.^10-14 Depending on the anticoagulant-preservative solution, packed RBCs can be maintained at 4°C for 42 days in sterile blood banks. Eight red blood cells alter during preservation.^14-20 Biconcave disc form loss, echinocytic spines, and microvesicle blebbing are seen.^21-23Chemical changes include glucose consumption, lactic acid accumulation, potassium loss, calcium gain, hemoglobin-bound NO loss, ATP and 2,3-diphosphoglycerate (DPG) decreases, pH drop, and lactate buildup^24-30. Functional changes include reduced oxygen delivery at conventional partial pressures, circulatory survival, and integrity.^31-33
The revised FDA criteria that 95 percent of RBC units have 1% hemolysis with 95 percent confidence at storage ends establishes the mean end-of-storage hemolysis at 0.35.15.16 Due to slower glycolytic enzyme activities at 1 to 6°C storage temperature, progressive acidification of the intracellular pH in the closed system of a bag, and reversible and irreversible oxidation of the active site of rate-limiting glycolytic enzymes like glyceraldehyde 3-phosphate dehydrogenase, high-energy phosphate compounds, especially 2,3-diphosphoglycerate (2,3-DPG), are gradually consumed during the first two weeks of storage. 17 Between storage weeks 2 and 3, ROS and oxygen saturation cause irreversible oxidation of purines, lipids, and proteins (carbonylation, nonenzymatic glycation, and thiol oxidation.^12,17-22 Transfusion following prolonged RBC storage is related to poor clinical outcomes in critically ill, cardiac surgery, and trauma patients, according to current studies.^18-22

Oxidation-reduction (Redox) processes, especially those involving reactive oxygen species (ROS), are at the core of practically every biochemical event that occurs within the body and in physiologically active surroundings, such as those found in preserved blood units.²³ The entire amount of oxidative stress in the environment is represented by these redox processes, which involve the transfer of electrons between oxidants and reductants.^24-27 The constant monitoring of these ubiquitous coupled reactions in blood, red blood cell (RBCs) units, and other biological fluids is known as ambient redox potential (RP). With several articles demonstrating oxidative damage as a significant factor in red cell storage lesions, this equilibrium may be very crucial in terms of the health and viability of RBCs^27-36 Blood storage is accompanied by the steady accumulation of a set of biochemical and morphological changes known collectively as the "storage lesion “that will decrease the efficiency of RBCs transfusion as a therapeutically process.

2. MATERIAL AND METHODS:

2.1. Blood Collection :

Jordanian healthy young individuals (males) donated twenty whole blood units (480±48ml). Provided informed consent was taken from each one. Participants were at least 20 years of age up to 50 years old and met all AABB and FDA requirements for blood donation. Whole blood was collected into citrate-phosphate-dextrose adenine anticoagulant (CPDA) that contains 50 ml preservative solution total (500ml) on each unit and stored the unit at 1 to 6°C according to standard procedures for 35 days.

2.2. Blood sample collection and laboratory procedures.

Blood samples were collected in the appropriate tube for each test at a different time of storage (zero, 7, 14, 21, and 35 days). Plasma was separated to perform potassium tests on Cobas c 311.2ml EDTA tube were used for hemoglobin, red blood cells count, and mean cell volume analysis on Automated hematology Sysmex XN1000. One mL whole blood was collected in ice from the 20 units at a different time of preservation (zero, 7, 14, 21, and 35 days) to perform the PH test on ROSH machine within thirty minutes. Two ml Eppendorf whole blood tube from each unit was taken at different time ranges: zero, 7, 14, 21, 28, and 35 days, then centrifuge (15000RPM for 15min) to separate the supernatant to perform antioxidant enzyme analysis including:

2.3. Human Glutathione Peroxidase (GPX1):

Kit ELISA, Sensitivity: 5.2ng/mL, Standard: 200, Range: 3.13–200. This kit's microtiter plate is pre-coated with GPX1 antibody. Standards or samples were added to microtiter plate wells then a biotin-conjugated Glutathione Peroxidase 1 antibody (GPX1. To assess the enzyme-substrate reaction, a sulphuric acid solution is added and the color change is determined at 450nm± 10nm spectrophotometrically. Comparing the samples' OD to the standard curve determines their GPX1 concentration.²⁷

2.4. Human Lipid Peroxide (LPO):

Kit ELISA, Sensitivity (0.058), Standard (10ng/mL), Range (0.16-10ng/mL). The microtiter plate in this kit is pre-coated with a Lipid Peroxide antibody. A biotin-conjugated LPO antibody is applied to microtiter plate wells with standards or samples. The enzyme-substrate reaction is stopped by adding sulphuric acid solution, and the color change is recorded at 450nm ± 10nm spectrophotometrically. The samples' LPO concentration is then calculated by comparing their OD to the standard curve.

2.5. Human Catalase (CAT):

ELISA Kit, sensitivity 0.124U/mL, standard 20U/mL, range 0.32-20U/mL. The kit's microtiter plate is pre-coated with CAT antibodies. A Catalase-specific biotin-conjugated antibody is applied to microtiter plate wells with standards or samples. The enzyme-substrate reaction is stopped by adding sulphuric acid solution, and the color change is recorded at 450nm±10nm spectrophotometrically. Comparing the samples' OD to the standard curve determines their CAT content.

2.6. Statistical analysis:

SPSS ver. 26.0 (SPSS Inc., Chicago, IL, USA) were used to perfume all statistical evaluations including Mean values, standard deviations for all parameters, and Comparisons between the result of different parameters at different days of sampling were done.^28,29

3. RESULTS AND DISCUSSION:

3.1. Results:

3.1.1. Descriptive statistics for the differences of number of selected parameters of RBCS storage in vivo:

The mean and standard deviation are shown in table 1 for the study sample is describes the differences of number of selected parameters from zero time to five weeks of RBCs storage in vivo. These parameters include: Red blood cell count (RBCs) count, Hemoglobin (Hb), potassium level, mean cell volume (MCV), PH, Catalase, glutathione peroxidase, and lipid peroxide. All the studied parameters showed significant difference at 35 days compared to zero time (P-Value < 0.05).

Table 1: The results of the laboratory tests at zero and 35 days of storage for the studied units.

	ZERO time		35 DAY		P-value
	Mean	S. D	Mean	S. D	P-value
PH	7.3900	0.024	6.5*	0.046	<0.001
RBCs	5.28	0.44	4.60*	0.36	<0.001
Hb	15.84	1.12	13.9*	0.77	<0.001
K	4.2305	0.39	29.8*	2.6	<0.001
MCV	88.2300	4.2	101.5*	5.08	<0.001
Catalase	3.4826	0.93	7.2*	2.28	<0.001
glutathione peroxidase	21.3171	6.8	41.3*	26.9	0.007
lipid peroxide	0.2055	0.13	0.33*	0.14	0.017

3.1.2. The correlation between Parameters of the oxidative stress and RBC’s storage time:

The pH levels declined from (7.39±0.02) to (6.54±0.04) within the storage period as shown in Figure 1. Numerical differences were calculated to quantify the deterioration rates of these metabolic markers, for which statistically significant differences were observed.

Figure 1: The intracellular PH at different time of storage.

Potassium:

Increased extracellular potassium is observed Due to the increased RBC hemolysis from storage. The mean extracellular K+ was increased from 4.23±0.39 at zero time to 29.86±2.68 after 35 days as shown in Figure 2.

Figure 2: The extracellular K+ at different time of storage.

MCV:

Statistically significant differences in the measured MCV were observed after 5 weeks of storage. The MCV was also increased from 88.23 ± 4.23 at the beginning of storage to 101.58 ± 5.08 at the end of storage (after 5 weeks) as appeared in Figure 3.

Figure 3: The mean MCV results at different time of storage.

Catalase, glutathione peroxidase and lipid peroxide:

Levels of Catalase, glutathione peroxidase and lipid peroxide were checked for storage lesion in the stored RBC. The results shown an increase in lipid peroxide, glutathione peroxidase, and Catalase activity as observed in 35 days of storage (Figure 4, 5, and 6 respectively).

Figure 4. The Lipid peroxidase results at different time of storage.

Figure 5. The Glutathione peroxidase results at different time of storage.

Figure 6. The Catalase results at different time of storage.

3.2. DISCUSSION:

Erythrocytes are a special type of therapeutic biological product that can only be provided by volunteers and cannot be manufactured by any other method under present technical conditions.³⁰Notably, The FDA, the National Institutes of Health and other organizations standards have shown that the preferential use of the stored RBCs is up to 42 days.^30-33. The most common procedures for storing erythrocytes are suspension in maintenance solution and refrigeration at 4±2°C for 35-42 days.³² Low temperatures are known to delay the metabolism of stored erythrocytes, and the maintenance solution provide nutritional support to the erythrocytes ³³ Europe and North America Current Quality Control (QC) recommendations advocate assessing blood product quality indices such as hemolysis, hematocrit, hemoglobin, and residual WBCs.^30,34-40. The standard storage buffer used for both RBC and whole blood is CPDA (Citrate, phosphate, dextrose and adenine), and SAGM (sodium, adenine, glucose and mannitol as components). each component of the buffer has different roles in preservations of the blood and its components. ^34,41 this study was aimed to evaluate the RBCs damage during the storage period in the blood bank before blood transfusion through measuring the alteration of number of different parameters.

This study agreed a previous study that approved those RBCs stored for an extended period of time (35 days or more) were subject to numerous changes in biochemical and biomechanical properties. The geometric characteristics of the cells are impaired and they become more rigid.^36,37,42 Their ability to change shape in response to mechanical stresses is further diminished, though this unique ability of RBCs to deform is essential for maintaining a healthy microvasculature.^36,38-43. It was observed in this study that the mean level of the extracellular K+ was increased from 4.23mmol/L at zero time to 29.86mmol/L after 35 days. Potassium ions increased significantly through to 35 days of storage. This result compares with previous studies in Portugal, Uganda, India and Nigeria which demonstrated that potassium significantly increased throughout the storage period.³⁷ Another study conducted in the western region of Kenya showed that the plasma potassium level increased to 20.14mmol/L at the conclusion of the fifth week which also approved our result.^36-39 This increasing Due to the elevation of RBCs hemolysis from storage. Blood preserved at 1 to 6 degrees Celsius slows cellular metabolism and energy requirements, rendering the sodium-potassium pump inactive and allowing potassium ions to escape the cell and sodium ions to enter across the semipermeable membrane.³⁹ Because of the potassium levels, blood transfusions beyond the first week of storage may be unsafe for patients. Even little fluctuations in potassium ion concentrations can be dangerous during severe renal disease, hence relatively fresh or cleaned red cells are advised.^40-43Based on the findings of this study, it is advised that potassium ion concentrations be monitored during blood storage for transfusion to improve blood safety.

Storage of RBCs resulted in slight swelling of the cells as measured by an increased mean corpuscular volume (MCV).⁴¹.This result was approved by our study which showed an MCV increasing from 88.2±4.2 to 101.6± 5.1. It was also previously documented that the higher MCV signifies a gradual increase in erythrocyte size due to extracellular sodium ion influx and accumulation in the cytosol.^31,42 The standard range for blood pH is 7.35-7.45; this study observed a decline in blood pH from 7.39 to 6.54 after 35 days of storage, which was comparable to an average drop of 0.85. This implies that the pH has dropped significantly below the usual range by the conclusion of the 35-day period. This result agreed with previous studies in Uganda, India, Ghana and Kenya which reported a significant pH decrease after 14 days of storage and further decreased significantly at 35 days of storage.³⁸ After storing for 14 days, lactate builds up and increases protons, which lowers pH and interferes with glycolytic metabolism.⁴³ The combination of an increased lactate level and a reciprocal decrease in pH has disastrous effects for blood recipients, particularly those who may be given numerous units of blood in a short period of time.⁴⁴This reduces blood potency and predisposes blood recipients to transfusion-related morbidity and death. The increase in pH directly leads to an increase in the activity of phosphofructokinase of erythrocytes, increased glucose consumption; the function of glycolysis was enhanced.^{17, 30}pH decrease is also connected to diphosphoglycerate depletion and decreased glycolytic activity.^2,45 This study suggests that transfusion of blood held for more than one week be done with caution.^38-42Considering the findings of this study, it is advised that pH levels be monitored during blood storage, particularly after the first week of storage, with the goal of enhancing blood safety.

In erythrocytes, nonconjugated polyunsaturated fatty acids with reactive methylene groups that are susceptible to hydrogen atom abstraction are prevalent.^43-46 The oxidation of these fatty acids by free radicals boosts malondialdehyde (MDA) levels, which are the final outcome of lipid peroxidation. MDA is a consequence of lipid peroxidation, which occurs when free radicals react with lipid and lipid derivative molecules.^45-47 Throughout the storage period, several factors may cause oxidant molecules such as superoxide anion (O₂), H₂O₂, and the hydroxyl radical (HO) to develop.^31,44,48 They have the ability to damage erythrocyte protein and lipid components.⁴⁶

Oxidative stress occurs in the cell when the formation of free radicals overwhelms the antioxidant defense system, resulting in oxidative damage to the cell.⁴¹Catalase, lipid peroxidase, and glutathione peroxidase are the most important antioxidant enzymes in human RBCs. A number of studies show that the major function of these enzymes in erythrocytes is to eliminate H₂O₂ and/or scavenge free radicals.^42,43The current investigation found that the concentration of these enzymes (Catalase, lipid peroxidase, and glutathione peroxidase) was significantly higher at 35 days of storage than at zero time. Catalase is a protein that catalyzes the conversion of hydrogen peroxide into less reactive gaseous oxygen and water molecules.^29,44-49 This indicates the breakdown of hydrogen peroxide, as catalase works best when H₂O₂concentrations are extremely high.⁴⁵ In a prior study done in the USA, the catalase activity at 42 days of storage compared to zero time showed no significant changes and a sharper fall compared with the other two enzymes.^41,46,48 Glutathione (GSH) is a redox active biomolecule that is essential for cellular and organism homeostasis. GSH reduces H₂O₂, a reactive oxygen species, to H₂O in a process catalyzed by glutathione peroxidase (GPX).³¹ The concentration of GPX was steadily raised during the storage period as it was appeared in this study, from 21.3 at zero time to 41.3 at 35 days. Glutathione Peroxidase enzyme activity decreased on days 7 and 29, respectively, according to another study (P value < 0.05), contradicting our findings^47-49 There was a noted rise in lipid peroxidation during the storage time. Our findings are consistent with their hypothesis that erythrocyte antioxidant systems would be impaired after prolonged storage in blood.⁵⁰

4. CONCLUSIONS:

Transfusion of RBCs is one of the most important and effective lifesaving treatments worldwide. Although there was marked biochemical and hematological changes in stored blood, this study revealed that preserved RBCs in the current storage media that preserved their parameters had an .acceptable quality for blood transfusion. This study was conducted with low count of RBCs units from males only. We therefore recommend further studies be done with different blood bags with larger units count from both genders to investigate their deterioratory propensities. Furthermore, investigate the role of antioxidants, either given to the donor before donation or added to the blood bag after red cell separation, in preventing oxidative damage to red cells during storage.

FUNDING:

The Deanship of Graduate Studies at Al-Ahliyya Amman University in Amman, Jordan, contributed funding for this study.

CONFLICT OF INTERESTS:

The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT:

The data underlying this article will be shared on reasonable request to the corresponding author.

AUTHOR CONTRIBUTIONS:

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article and revising it critically for important intellectual content; agreed to submit to the current journal; and agree to be accountable for all aspects of the work.

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Received on 08.02.2024 Modified on 22.05.2024

Research J. Pharm. and Tech 2024; 17(9):4304-4310.

DOI: 10.52711/0974-360X.2024.00665